Functions of Dicer, Drosha and miRNAs in the skin

描述（由申请人提供）：microRNA在表皮和毛囊形态发生和稳态中的功能是皮肤生物学的一个令人兴奋的新领域，对我们理解皮肤和毛囊功能以及皮肤疾病（包括脱发、皮肤肿瘤发生和伤口愈合缺陷）具有重要意义。本文提出的实验使用体内和体外技术来更精确地定义miRNAs在皮肤稳态中的整体作用，并开始鉴定对正常皮肤功能至关重要的特定miRNAs及其靶基因。因此，我们相信我们的研究将对我们理解皮肤生物学具有重大意义，并有可能最终揭示从脱发到皮肤肿瘤等一系列疾病的治疗靶点。最近发现的microRNAs（miRNAs）的转录后调控揭示了一个以前未知的机制，基因表达的控制和完善。非蛋白质编码的miRNA前体（pri-miRNA）从核基因转录并形成独特的茎环结构，其被核酶Drosha及其辅因子DGCR 8特异性识别和切割以形成pre-miRNA。前体miRNA从细胞核输出并通过Dicer酶加工成约22个核苷酸的成熟miRNA。miRNA组装成RISC复合物，其通过抑制在其3'非编码区中含有部分互补序列的靶mRNA的翻译和/或使其不稳定来调节基因表达。在哺乳动物中已经鉴定出数百种miRNA，其中至少33种在皮肤中高度表达。独特的小鼠Dicer基因的组成性表皮缺失导致成熟miRNA的丢失和多种表型，包括：毛囊的形态发生和维持失败;毛囊干细胞标志物表达的缺失;表皮-真皮信号传导中断;毛囊真皮乳头外翻;以及表皮过度增殖和细胞凋亡。基于这些表型，我们假设Dicer是出生后维持毛囊生长和表皮稳态所必需的，并且它部分地在毛囊干细胞内起作用。我们进一步假设表皮Dicer突变表型由miRNA缺失引起。为了验证这些假设，我们将：（i）诱导性地删除出生后皮肤中的表皮Dicer;（ii）删除毛囊干细胞中特异性的Dicer;（iii）确定另一种必需的miRNA加工途径组分的表皮耗竭是否复制了表皮Dicer缺失的影响;（iv）表征在不同皮肤区室中表达的miRNA，并开始鉴定其靶mRNA。

DESCRIPTION (provided by applicant): microRNA function in epidermal and hair follicle morphogenesis and homeostasis is an exciting new area of skin biology that has major implications for our understanding of skin and hair follicle function and skin disorders including alopecias, skin tumorigenesis, and wound healing defects. The experiments proposed here use in vivo and in vitro techniques to more precisely define the global roles of miRNAs in skin homeostasis, and to begin to identify specific miRNAs and their target genes that are critical for normal skin function. As such we believe that our studies will have major significance for our understanding of skin biology and have the potential to ultimately reveal therapeutic targets for a range of diseases from alopecias to skin tumors. The recent discovery of post-transcriptional regulation by microRNAs (miRNAs) revealed a previously unsuspected mechanism by which gene expression is controlled and refined. Non-protein coding miRNA precursors (pri-miRNAs) are transcribed from nuclear genes and form distinctive stem-loop structures that are specifically recognized and cleaved by the nuclear enzyme Drosha and its cofactor DGCR8 to form pre-miRNAs. Pre-miRNAs are exported from the nucleus and processed to approximately 22 nucleotide mature miRNA by the Dicer enzyme. miRNAs are assembled into the RISC complex which regulates gene expression by inhibiting the translation of, and/or destabilizing, target mRNAs that contain partially complementary sequences in their 3' non-coding regions. Several hundred miRNAs have been identified in mammals, and at least 33 are highly expressed in the skin. Constitutive epidermal deletion of the unique mouse Dicer gene results in loss of mature miRNAs and multiple phenotypes including: failure of morphogenesis and maintenance of hair follicles; absence of expression of hair follicle stem cell markers; disrupted epidermal-dermal signaling; evagination of hair follicle dermal papillae; and epidermal hyperproliferation and apoptosis. Based on these phenotypes we hypothesize that Dicer is required postnatally for maintaining hair follicle growth and epidermal homeostasis, and that it functions, in part, within hair follicle stem cells. We further hypothesize that epidermal Dicer mutant phenotypes result from miRNA depletion. To test these hypotheses, we will: (i) inducibly delete epidermal Dicer in postnatal skin; (ii) delete Dicer specifically within hair follicle stem cells; (iii) determine whether epidermal depletion of another essential miRNA processing pathway component replicates the effects of loss of epidermal Dicer; (iv) characterize miRNAs expressed in different skin compartments and begin to identify their target mRNAs.